The present invention relates to a method of producing value-added milk by the incorporation of specific DNA sequence(s) into the cells of the mammary gland. The term xe2x80x9cvalue-added milkxe2x80x9d is meant to mean milk containing a valuable compound, such as a pharmaceutical, as well as milk with a changed composition so that the market value of the milk is enhanced.
This invention is based upon a technique to transfer a gene, and related non-translated control sequences, into the secretory cells of the mammary gland to produce new compounds in milk and/or to alter the milk composition.
An example of a compound produced into milk by this method would be a pharmaceutical which cannot be manufactured in a biologically active state. This invention provides an alternative method of producing such pharmaceuticals into milk using the synthetic capabilities of the mammary epithelial cell.
Mammals produce copious amounts of complex proteins into milk to provide nutrition for their young. To harness this capability, the DNA of, for example a valuable pharmaceutical, can be inserted into a mammary epithelial cell and this cell then will produce the active, pharmaceutical compound into the secreted milk. In a cow, the udder will act as a receptacle to hold the milk until it can be collected. The milk containing the added compound can be processed to extract and purify this pharmaceutical compound for subsequent sale, possibly to the medical and/or veterinary communities, or the milk could be consumed directly as a therapeutic agent.
At present there are two basic techniques that can be used to produce value-added milk. The first is to create a transgenic animal by microinjection or transfection of foreign DNA into an ovum or a fertilized egg. Incorporation of DNA at this stage in development generally results in a transgenic animal which carries the inserted DNA in every cell.
There are positive and negative aspects of producing a transgenic animal which expresses foreign proteins in its milk. A positive point is that a single founder animal can create a population of transgenic animals by natural reproduction. However, progenies do not always produce the exogenous protein at the same level as the original animal. Negative aspects include the technically difficult procedures required to produce the animal and the long time between adding the foreign DNA and harvesting the exogenous protein. In addition, the presence of even very small amounts of an active pharmaceutical in every tissue may be detrimental to the health of the animal.
An alternative method is to produce value-added milk by adding the desired DNA only to the cells of the mammary gland of the animal. This results in a trans-somatic animal (or chimera) which contains the inserted DNA essentially in only one tissue, the mammary gland.
Trans-somatic animals have the advantage that they can be produced with less technical difficulty. They also can be produced quickly so that there is a period of only weeks to months between adding the foreign DNA and harvesting the exogenous protein. Moreover, since only one tissue contains the added DNA and produces the resulting compound, health risks to the trans-somatic animal are reduced. Although the DNA is not passed on to the progeny, this is compensated for by the ease and speed with which a trans-somatic animal can be produced.
A trans-somatic goat which expresses human growth hormone (hGH) into milk has been produced by Archer et al. (1994). In Archer the viral-derived particles were infused into the mammary gland for approximately every two days for two weeks. The levels of the compound, human growth hormone, which was used as an example, were very low and approached background levels after the first day. Also Gould et al. (U.S. Pat. No. 5,215,904) described a method for increasing the rate of mitosis of mammary epithelial cells and then exposing these cells to viral particles for integration of the desired DNA into the epithelial cell.
To produce either a trans-somatic or a transgenic animal, exogenous DNA must pass through the exterior cell membrane. Eukaryotic cells have evolved a membrane which is impervious to most substances including heterologous DNA. Numerous techniques have been developed to bypass this barrier. These include:
electroporation,
carrier lipids (liposomes, negative, positive or neutral charged vesicles),
mechanical wounding of cells including microinjection, liquid or air-jet pressure and scrape loading,
use of particles composed partially or wholly of viral proteins.
Methods which have successfully produced trans-somatic animals include:
1) Arterial injection of DNA carried in liposomes (small lipid vesicles) was used to produce a trans-somatic mouse (Thierry et al. 1995). This technique can be adapted to deliver DNA to the mammary gland by injection into the major artery serving the mammary gland but circulation of the blood carrying the DNA can result in transfection of multiple tissues.
2) Direct injection of the DNA into tissues was used successfully to add foreign DNA to muscle and other tissues (Furth et al. 1992). This technique can be adapted to inject virus-like particles, carrying the foreign DNA, directly into the tissue of the udder.
3) Use of viral-derived particles carrying DNA coding for human growth hormone (hGH) were infused through the teat canal, for example by Archer et al. (1994). This resulted in production of trans-somatic goats which expressed hGH into the milk.
Viruses reproduce within cells and therefore have evolved a technique to by-pass the protective cell membrane to deliver the viral genome (DNA) into a host cell. To enter a cell, protein(s) of the outer viral shells first bind to receptors on the cell surface and then the virus is internalized.
The method used by Archer involves transfecting a cell line with DNA coding for various, but not all, proteins of a virus. This cell line, called a xe2x80x9cpackaging cell linexe2x80x9d, will produce empty virus shells which can bind to receptors on the host cell membrane. When heterologous DNA, coding for a pharmaceutical or other milk modification, is transfected into the packaging cell line, this DNA will be packaged into the viral-derived particle. When the viral-derived particle comes in contact with a milk-producing cell, the viral proteins of the shell ensure that the heterologous DNA is carried into the cell. Other viral proteins, associated with the particle, integrate the heterologous DNA into the genome of the host cell so that the protein encoded by the DNA can be expressed. In this method the viral-derived particles are used to introduce the heterologous DNA into the mammary gland.
The trans-somatic methods of the prior art offer advantages over the transgenic method; however the very low levels of foreign protein in the milk of the trans-somatic animal have limited the commercial success of these methods.
Thus the present invention is directed to methods of improving the yield of the foreign protein in the milk of a trans-somatic animal.
The present invention relates to a method of producing valuable compounds into milk and/or changing the composition of milk so as to enhance its properties and/or its marketability. More specifically the present invention relates to the addition of specific DNA sequences, including non-translated regulatory sequences, to the cells of the mammary gland and the subsequent expression of compound(s) encoded by that DNA into the milk. Other DNA sequences that enhance the efficiency of production of the compound, enhance the stability of the compound, or result in biological activity of the compound can also be added to the mammary epithelial cells either at the same time or at a different time.
The novel method of the present invention involves the use of viral-derived particles and packaging cells which produce these particles for infusion into the mammary gland through the teat canal. The packaging cells will attach and survive for a period of time within the mammary gland. While the cells are viable, they will supply a continuous source of viral-derived particles. These viral-derived particles trans-infect only dividing cells and are destroyed relatively quickly in the mammary gland. Thus a continuous supply of viral-derived particles from the packaging cells present in the mammary gland will ensure that viral-derived particles are present and can trans-infect the mammary epithelial cells whenever they divide.
The packaging cells and viral-derived particles can be from retroviruses and from non-retroviruses. Most retroviral-derived particles trans-infect only dividing cells. Non-retroviral particles such as those from adenovirus, Epstein-Barr virus, or other viruses trans-infect non-dividing cells. Thus a mixture of the two types of particles, and the associated packaging cells if needed, will ensure delivery of the packaged DNA to the maximum number of cells. Moreover, DNA can be packaged into viral-derived particles in vitro and these can be used for trans-infection as well.
This method can be used alone or in combination with other novel methods designed to ensure that the viral-derived particles are correctly positioned to trans-infect the mammary epithelial cells. Increasing the trans-infection of the mammary epithelial cells with the viral particles will result in a higher concentration of the valuable compound in the milk along with possible other compounds produced from the inserted DNA that will enhance the efficient production, stability or activity of the compound.
Thus according to the present invention there is provided a method of producing a trans-somatic mammal, wherein said method provides the incorporation of a DNA sequence into the secretory cells of the mammary gland to alter the composition of the milk, comprising the steps of: providing a vector containing a DNA sequence encoding a valuable compound; packaging said vector into a cell line; preparing a solution comprising the packaged vector and cell line producing said packaged vector; and delivering said solution into the mammary gland to allow the incorporation of the DNA into the secretory cells of the mammary gland.